Hepadnaviruses

1
Chapter 18

• Hepadnaviruses
• (and other reverse-transcribing
• DNA viruses)

2
Introduction

• Called hepadnavirus because cause hepatitis and
  are DNA viruses
• Hepatitis B virus (HBV) some infect mammals
  like woodchuck HBV and some infect birds like
  heron HBV
• HBV in humans is agent of disease and death
  whereas duck HBV is non-pathogenic in natural
  host
• Interesting points
• very small genome used with economy to encode
  all proteins and control expression
• pararetrovirus DNA replicated via a RNA
  intermediate reverse transcription
• also found in pant viruses like the cauliflower
  mosaic virus

3
Importance of HBV

• 400 million infected but just estimate
• Most cases in Asia and Africa, appear high in
  Canada and Greenland but that is because
  population is low
• Virus present in blood and semen of infected
  individuals
• modes parallel HIV sexually, reuse of syringe
  and needles (8 million cases/year)
• gt50 million cases/year babies from infected
  mothers

4
HBV Infection

• Many infections are mild or asymptomatic
  especially in kids but likely to be persistent
  90-95 carriers if infection as newborns whereas
  1-10 if infected as adult
• Persistent infection may be fine but some will
  get severe hepatitis which may lead to cirrhosis
  and eventually liver cancer
• 1 million deaths/year

5
HBV Virion

• Roughly spherical
• Main components envelope enclosing capsid with
  DNA and P (polymerase) protein inside
• DNA is dsDNA 1 which is incomplete part is
  ss/part ds short sequence is triple stranded as
  result of complimentary sequence at 5 ends
• looks circular
• 3.2 kb(p)
• at each 5 end is covalently linked molecule
  capped RNA on short strand and P protein on long
  strand

6
P Protein

• Polymerase (P) protein DNA-dependent DNA pol
• At least 1 complete molecule of P per virion
• N end of P is a terminal protein domain
  separated by spacer from the reverse
  transcriptase activity
• C end contains the RNase H domain

7
Capsid

• Icosahedral symmetry
• Has holes and short spikes protruding from the
  structure
• Constructed of dimers of C (core) protein which
  is mostly ? helices unlike other capsid proteins
• C end is highly basic due to large number of Arg
  residues that bind the viral genome

8
Envelope

• Has 3 protein species
• small (S) most abundant on surface
• medium (M)
• large (L)
• M and L are longer versions of S
• Surface regions constitute an Ag as Hepatitis B
  surface Ag HBsAg
• Each protein has 1 or more glycosylation sites
• Viral attachment near the N of L protein
• 50 of L have N to outside of virion
• remaining N have the N of L protein on inside
  bound to the capsid

9
Non-Infectious Particles

• Unusual large quantities of non-infectious
  particles in the blood
• Composed of lipid and envelope proteins but no
  nucleocapsid
• spherical or filamentous of varying lengths
• Particles (infectious and non-infectious) are
  more numerous in blood than liver with
  non-infectious in the majority
• not understood why present but may be a decoy for
  Ab made to envelope

10
Soluble Virus Protein

• Protein found in the blood with virions and
  non-infectious particles
• Known as e Ag HBeAg
• similar to C protein with 10 extra amino acids at
  the N end and missing 34 amino acids at the C
  end
• unknown function

11
HBV Genome

• 3.2 kb not smallest
• 4 ORFs that make 7 proteins large amount of
  coding information in small genome use every
  nucleotide and more than ½ genome in 2 ORF
• P ORF (80 genome) overlaps C and X ORFs
• S ORF is within P ORF
• Heavily dependent on host cell proteins to play a
  role in HBV replication enzymes, TF and
  chaperones

12
Genome (continued)

• Increase coding capacity by expressing L protein
  in 2 conformations
• 1 functions as virus attachment
• 1 functions as binding virion envelope to capsid
• Promoters are within the coding sequence
• Genome has directed repeats of 11 nucleotides
  DR1 and DR2
• Expression of pre-S-pre S2-S region 3 proteins
• S region S protein
• pre S2-S M protein
• pre S-pre S2-S region L protein
• Pre C-C region gives us 2 proteins

13
HBV Genetic Groups

• 8 genetic groups A thru H
• Human viruses related to similar viruses in
  primate species
• Restricted geographically
• genotype A is in Northern Europe
• genotype B and C is in Asia

14
HBV Replication

• Hepatocyte is the host cell
• can use liver cells in culture but unable to
  infect liver tumor cells using the virion but can
  transfect the DNA into these cells and get
  infectious viruses
• Can infect cells with woodchuck or duck virions
  so most of what we know are from these viruses or
  DNA transfection studies

15
Attachment

• Dont know the receptor that is used by virus
• Possibilities are IgA and annexin V
• Binds to the virus attachment site on the L
  protein

16
Entry

• Endocytosis
• Nucleocapsid is released from endosome by
  membrane fusion
• Nucleocapsid is seen entering the nucleus can
  pass thru nuclear pore but not sure whether
  uncoating of genome is within the pore or in
  genome is released into nucleus with capsid
  staying on the outside

17
Genome Circularization

• Once genome enters nucleus it circularizes
• Covalently bound P protein is removed from the 5
  end of (-) strand, removes the 3rd strand of
  triple-stranded region
• RNA is removed from 5 end of the () strand
• DNA synthesis at 3 end makes entire molecule
  double stranded
• End of each strand is ligated forming a
  covalently closed circular DNA cccDNA
• not sure modification to virus genome by host
  enzymes or whether P protein plays a role
• DNA not replicated in nucleus brought into
  nucleus later

18
Transcription

• cccDNA is template for transcription
• HBV genome has 4 promoters upstream of pre-S1,
  pre-S2 (only TATA box), X and pre-C regions
• at least 2 are specific to liver cells
• some TF are liver cell proteins may be why
  specific to liver and not other cells
• Rate of transcription from 4 promoters is
  controlled by cellular TF binding to 2 HBV
  enhancers
• X protein is thought to be atypical TF as doesnt
  bind DNA

19
Transcription (continued)

• Cellular RNA pol II makes 4 size classes of RNA
• All are capped at 5 end and polyA tail at 3 end
• use TATAAA signal at the 3 end so share a common
  3 end
• 3.5 kb mRNAs are longer than genome part of
  genome is trancribed twice
• use direct terminal repeats so RNA pol must
  ignore polyA signal on first pass
• in duck HBV controlled by DNA sequence PET,
  positive effector of transcription allow RNA
  pol to continue thru poly A signal
• PET susspresses NET, negative effector of
  transcription which is needed for transcription
• remove NET, RNA pol makes very long transcripts

20
Translation

• Translation of 6 transcripts
• RNA 3.5 kb 2 subsets that differ slightly in
  size
• shorter subset has not start codon in pre-C
  sequence so acts as mRNA for C and P proteins
• C is ORF upstream and P is ORF downstream
• ribosome start translation at C start codon
• at least 3 start codons before P start codon
• ribosome by-passes by leaky scanning so make
  more capsid than polymerase proteins
• shorter subset also acts as pre-genome
• HBeAg translated from longer subset, start codon
  for pre-C sequence secreted from cell after
  translation
• 2 subsets for 2.1 kb mRNA
• M protein is translated from the longest subset
• S protein from the shorter subset

21
Post-Translational Modifications

• Some envelope proteins become glycosylated
• L is myristylated at N
• not necessary for assembly but if made without,
  virions are not infective
• L protein N are on cytoplasmic side of ER but
  after translation about 50 of N are moved thru
  membrane into lumen of ER
• thought to be aided by heat shock protein Hsc70

22
Nucleocapsid Assembly

• C protein forms dimer and assemble into capsids
• can self-assemble in E coli that express HBV C
  gene
• Molecule of P along with cell proteins bind 3.5
  kb RNA to function as pre-genome RNA

23
Pre-Genome RNA

• P binds at sequence called ? (within terminal
  repeat at both ends) with a high degree of 2
  structure
• P only binds to 5 site and makes packaging
  signal incorporating pre-genome RNA into the
  capsid

24
Genome Synthesis

• Reverse transcriptase domain of P proteins
  carries out DNA synthesis and terminal domain of
  P protein (Tyr OH) acts as primer initiate (-)
  DNA strand synthesis
• not sure whether terminal protein is cleaved or
  not after initiation
• Pre-genome RNA is template for DNA synthesis
• 4 nt (-) DNA is synthesized and transferred to
  complementary sequence in DR1 near 3 end of
  pre-genome continues to the 5 end of
  pre-genome template
• RNase H activity removes RNA from RNA-DNA duplex
• removes all but the last 15-18 nt including the
  cap that is used as primer for DNA () strand
  synthesis

25
(No Transcript)
26
Assembly (continued)

• During ()DNA synthesis nucleocapsid migrates
  to nucleus to increase the amount of cccDNA or
  undergo maturation event to enable budding thru
  membrane
• DNA synthesis ceases on budding cut off from nt
  pool and () DNA strand is incomplete

27
Exit

• Membrane for budding is between RER and Golgi as
  budding occurs copies of L protein with N on
  cytoplasmic surface binds capsid
• Virions move to cell surface and released
• thought to occur in vesicles that fuse to plasma
  membrane, releasing by exocytosis
• non-infectious spheres and filaments also
  released
• Hepatocytes are not killed - many survive for
  many months
• Cannot see CPE in cell culture
• Damage to liver cells not caused by virus but
  rather the hosts immune system

28

• Still need additional information to fill in the
  gaps

29
Prevention

• Original vaccine made with non-infectious
  particles harvested from HBV carriers
• Now use recombinant yeast containing gene for S
  protein
• vaccination can reduce the number of carriers

30
Treatment

• Treat with ? interferon doesnt eliminate virus
  but results in significant reduction in viremia
  in 20-30 of cases
• side effects to interferon is influenza like
  symptoms, weight loss must weigh benefit vs
  side effects
• Lamivudine nucleoside analog improvement over
  ? interferon
• decreases viral replication with minimal side
  effects, oral vs injection and cheaper
• long term use may develop resistance but can use
  another nucleoside analog such as adefovir

31
Other Reverse-Transcribing DNA Viruses

• Some plant viruses with dsDNA replicate with an
  RNA intermediate
• Caulimoviridae
• cauliflower mosaic virus isometric virus
• rice tungo bacilliform virus rod shaped